Analysis module 4: 16S vs ORV outcome RoVI study — 02 November, 2020

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Sample summary

Alpha diversity

Beta diversity

Differential taxa

Longitudinal abundance comparisons

Sample summary

Input data: infant stools (BS1, BS2, BS3, and BS5) and maternal stool (MS1)

  • n samples = 2138
  • n taxa: genera = 490; variants = 6282
  • n taxa (rarefied): genera = 455; variants = 6143

Statistical methods for mixed-effects models

  • Linear mixed-effects models fit with family ID as a random effect
  • Association with ORV outcome determined by comparing fit of model with country/age vs model with age alone

Alpha diversity

Column

Genus-level

Seroconversion

Longitudinal plots

Statistics

                  Shannon log(Observed)
India              0.0099        0.0013
India (exposed)    0.3155        0.2959
India (unexposed)  0.0036        0.0001
Malawi             0.5418        0.5263
UK                 0.8155        0.5826

Post-ORV IgA

Dose 1 alpha diversity (week 6/8)

Correlation coefficients values
                India India_exposed India_unexposed  Malawi      UK
Shannon       -0.0194        0.0807         -0.1688 -0.3269 -0.0364
log(richness) -0.0538        0.0569         -0.1795 -0.2933 -0.0929
P values
               India India_exposed India_unexposed Malawi     UK
Shannon       0.7429        0.3201          0.0530 0.0074 0.8061
log(richness) 0.3640        0.4834          0.0394 0.0169 0.5300

Dose 2 alpha diversity (week 10/12)

Correlation coefficients values
                India India_exposed India_unexposed  Malawi      UK
Shannon       -0.0476       -0.0429         -0.0432  0.0767 -0.0630
log(richness) -0.0483        0.0600         -0.0950 -0.0380  0.0067
P values
               India India_exposed India_unexposed Malawi     UK
Shannon       0.4272        0.5903          0.6393 0.5852 0.6670
log(richness) 0.4198        0.4507          0.3022 0.7870 0.9635

Dose 1 shedding

Longitudinal plots

Statistics

                  Shannon log(Observed)
India              0.1704        0.1318
India (exposed)    0.8656        0.6844
India (unexposed)  0.2384        0.0572
Malawi             0.2673        0.1553

Shedding after either dose

Longitudinal plots

Statistics

                  Shannon log(Observed)
India              0.3228        0.1297
India (exposed)    0.9722        0.6468
India (unexposed)  0.3455        0.1966
Malawi             0.9498        0.4796

Neonatal infection

Longitudinal plots

Statistics

      Shannon log(Observed)
India  0.4005        0.5271

Cross-sectional comparisons

See Module 5 for full univariate and multivariate analyses.

RSV-level

Seroconversion

Longitudinal plots

Statistics

                  Shannon_rsv log(Observed_rsv)
India                  0.0182            0.0044
India (exposed)        0.2923            0.3252
India (unexposed)      0.0156            0.0012
Malawi                 0.9276            0.8659
UK                     0.9307            0.5172

Post-ORV IgA

Dose 1 alpha diversity (week 6/8)

Correlation coefficients values
                India India_exposed India_unexposed  Malawi      UK
Shannon        0.0032        0.0729         -0.1138 -0.1374 -0.1349
log(richness) -0.0555        0.0416         -0.2035 -0.1915 -0.0843
P values
               India India_exposed India_unexposed Malawi     UK
Shannon       0.9568        0.3690          0.1940 0.2711 0.3608
log(richness) 0.3487        0.6083          0.0192 0.1235 0.5689

Dose 2 alpha diversity (week 10/12)

Correlation coefficients values
                India India_exposed India_unexposed  Malawi      UK
Shannon       -0.0254       -0.0476          0.0746  0.0942 -0.0103
log(richness) -0.0398        0.0065         -0.0616 -0.0251 -0.0544
P values
               India India_exposed India_unexposed Malawi     UK
Shannon       0.6712        0.5502          0.4181 0.5023 0.9438
log(richness) 0.5062        0.9349          0.5036 0.8583 0.7107

Dose 1 shedding

Longitudinal plots

Statistics

                  Shannon_rsv log(Observed_rsv)
India                  0.5308            0.3872
India (exposed)        0.8026            0.6590
India (unexposed)      0.6945            0.2629
Malawi                 0.1322            0.1664

Shedding after either dose

Longitudinal plots

Statistics

                  Shannon_rsv log(Observed_rsv)
India                  0.9686            0.3633
India (exposed)        0.8933            0.6198
India (unexposed)      0.9022            0.6262
Malawi                 0.7173            0.9673

Neonatal infection

Longitudinal plots

Statistics

      Shannon_rsv log(Observed_rsv)
India      0.8768              0.64

Beta diversity

Column

Genus-level

Seroconversion

Bray-Curtis distances (weighted)

Bray-Curtis distances (unweighted)

RSV-level

Seroconversion

Bray-Curtis distances (weighted)

Bray-Curtis distances (unweighted)

Differential taxa

Column

Genus-level

Seroconversion

Post-ORV IgA concentration

Dose 1 shedding

Shedding after either dose

Neonatal exposure

RSV-level

Seroconversion

Post-ORV IgA concentration

Dose 1 shedding

Shedding after either dose

Neonatal exposure

Longitudinal abundance comparisons

Immunogenicity

Number of infants/taxa included in longitudinal models:

  • India (full), n infants: 305; n samples: 1140; n taxa: 25
  • India (exposed), n infants: 166; n samples: 625; n taxa: 26
  • India (unexposed), n infants: 138; n samples: 511; n taxa: 24
  • Malawi, n infants: 96; n samples: 263; n taxa: 41
  • UK, n infants: 51; n samples: 197; n taxa: 25

India

          
           negatively correlated ns positively correlated
  age                          6 12                     7
  IgA                          0 25                     0
  seroconv                     0 25                     0

Summary statistics

[1] "No significant differences according to ORV outcome"

India (exposed)

          
           negatively correlated ns positively correlated
  age                          7 11                     8
  IgA                          1 25                     0
  seroconv                     0 26                     0

Summary statistics

           taxon Estimate Std.Error   padj comparison  mean prev
59 Lactobacillus   -0.181     0.055 0.0364        IgA 3.685 62.7

India (unexposed)

          
           negatively correlated ns positively correlated
  age                          6 10                     8
  IgA                          0 24                     0
  seroconv                     0 24                     0

Summary statistics

[1] "No significant differences according to ORV outcome"

Malawi

          
           negatively correlated ns positively correlated
  age                         12 14                    15
  IgA                          0 41                     0
  seroconv                     0 41                     0

Summary statistics

[1] "No significant differences according to ORV outcome"

UK

          
           negatively correlated ns positively correlated
  age                         13  8                     4
  IgA                          0 24                     0
  seroconv                     0 25                     0
                  taxon Estimate Std.Error         padj comparison
2  Escherichia/Shigella    0.045     0.013 1.727273e-03        age
3         Streptococcus   -0.119     0.024 5.312500e-06        age
4        Staphylococcus   -0.435     0.025 1.825000e-35        age
7         Lactobacillus   -0.068     0.035 7.812500e-02        age
8           Collinsella   -0.024     0.013 7.941176e-02        age
9           Haemophilus   -0.226     0.023 1.125000e-16        age
10           Klebsiella   -0.156     0.020 3.125000e-12        age
11         Enterococcus   -0.116     0.024 1.194444e-05        age
14              Gemella   -0.120     0.020 3.250000e-08        age
15         Enterobacter   -0.050     0.017 7.142857e-03        age
16      Parabacteroides   -0.051     0.016 2.500000e-03        age
17    Corynebacterium_1   -0.164     0.023 1.666667e-10        age
20       Granulicatella   -0.063     0.015 1.225000e-04        age
22          Actinomyces   -0.234     0.032 5.500000e-11        age
23          Eggerthella    0.146     0.018 1.916667e-12        age
24       Negativicoccus    0.058     0.021 1.016667e-02        age
25           Prevotella    0.256     0.078 2.500000e-03        age

Summary statistics

[1] "No significant differences according to ORV outcome"

Dose 1 shedding

Number of infants/taxa included in longitudinal models:

  • India (full), n infants: 305; n samples: 859; n taxa: 23
  • India (exposed), n infants: 164; n samples: 459; n taxa: 24
  • India (unexposed), n infants: 138; n samples: 391; n taxa: 22
  • Malawi, n infants: 89; n samples: 189; n taxa: 43
  • UK, n infants: 60; n samples: 171; n taxa: 21

India

       
        ns
  dose1 23

Summary statistics

[1] "No significant differences according to ORV outcome"

India (exposed)

       
        ns
  dose1 24

Summary statistics

[1] "No significant differences according to ORV outcome"

India (unexposed)

       
        ns
  dose1 22

Summary statistics

[1] "No significant differences according to ORV outcome"

Malawi

       
        negatively correlated ns
  dose1                     1 41

Summary statistics

        taxon Estimate Std.Error    padj comparison mean prev
28 Sutterella   -1.418     0.369 0.00966      dose1 1.28 43.9

Shedding after either dose

Number of infants/taxa included in longitudinal models:

  • India (full), n infants: 304; n samples: 856; n taxa: 23
  • India (exposed), n infants: 164; n samples: 459; n taxa: 24
  • India (unexposed), n infants: 137; n samples: 388; n taxa: 22
  • Malawi, n infants: 64; n samples: 135; n taxa: 42
  • UK, n infants: 56; n samples: 160; n taxa: 22

India

        
         negatively correlated ns
  either                     2 21

Summary statistics

               taxon Estimate Std.Error    padj comparison  mean prev
17 Corynebacterium_1   -0.342     0.110 0.02415     either 0.022 65.5
22    Dolosigranulum   -1.069     0.339 0.02415     either 0.012 35.4

India (exposed)

        
         ns
  either 24

Summary statistics

[1] "No significant differences according to ORV outcome"

India (unexposed)

        
         negatively correlated ns
  either                     1 21

Summary statistics

               taxon Estimate Std.Error   padj comparison  mean prev
17 Corynebacterium_1   -0.769     0.246 0.0462     either 0.024 66.8

Malawi

        
         negatively correlated ns positively correlated
  either                     2 36                     4

Summary statistics

                   taxon Estimate Std.Error  padj comparison  mean prev
9            Haemophilus    1.430     0.461 0.084     either 0.404 40.0
10            Klebsiella    1.571     0.611 0.084     either 3.946 70.4
14               Gemella    0.919     0.351 0.084     either 0.029 64.4
26 Phascolarctobacterium    1.769     0.644 0.084     either 0.169 27.4
31          Holdemanella   -1.701     0.569 0.084     either 0.172 28.1
36            Lawsonella   -2.512     0.933 0.084     either 0.007 22.2

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